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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: Toxicity Study by Oral Gavage Administration to Sexually Mature Male CD Rats for 5 Days
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Between 4 December 2008 and 7 July 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accpted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The systemic toxic potential and testicular and epididymal toxicity of Bourgeonal (Industrial chemicals), to Crl:CD® (SD)IGS BR rats by oral gavage administration was assessed over a period of 5 days. Each test material was administered to three groups each comprising six sexually mature male CD rats that received treatment at doses of 25, 100 or 250 mg/kg/day. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose (5 mL/kg). A further group comprising 6 male rats was included in the study to act as a positive Control group and received the substance, Lilial, at a treatment dose of 250 mg/kg/day.
GLP compliance:
yes (incl. certificate)
Remarks:
(Inspection: 17-19 February 2009 Signature: 1 May 2009)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name : Bourgeonal
CAS # : 18127-01-0
Description : Clear colourless liquid
Storage conditions : At ambient temperature under nitrogen.
Batch number : VE000010051
Date of receipt : 20 November 2008
Expiry date : 4 August 2009
Purity : 98.4%

Test animals

Species:
rat
Strain:
other: Crl:CD® (SD)IGS BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Charles River (UK) Ltd.

- Age at study initiation:
12 weeks

- Weight at study initiation:
380 to 473 g.

- Fasting period before study:
Not specified.

- Housing:
Animals were housed inside a barriered rodent facility (Building 8, Room 08). The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms. Before the study the room was cleaned and disinfected.

- Diet (e.g. ad libitum):
The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet), except when urine was being collected on Day 5. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

- Water (e.g. ad libitum):
Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.

- Acclimation period:
14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C

- Humidity (%): 40 to 70%

- Air changes (per hr):
Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.

- Photoperiod (hrs dark / hrs light):
Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours, except on Day 1 of treatment when the light cycle was overridden on 3 occasions to accommodate technical procedures required by the Study Protocol.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
n/a
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical procedure - Bourgeonal

High performance liquid chromatograph (HPLC): Comprising any suitable pump, autosampler and UV detector with sufficient precision and sensitivity

Chromatography data handling: Waters Empower 2

Balances fitted with printers: Capability of weighing to 5 or 6 decimal places General laboratory apparatus and glassware.

Control vehicle: Corn oil

Methanol: HPLC solvent

Tetrahydrofuran (THF): HPLC solvent

Water: Reverse osmosis

Mobile phase: Methanol / water (65/35 v/v).

Analytical column: Waters Symmetry C18, 5 μm, 150 × 3.9 mm

Column temperature: Ambient

Flow rate: 1.0 mL/minute

Detector wavelength: 210 nm

Injection volume: 10 μL

Approximate retention time: 11.6 – 14.2 minutes

The analytical procedurse were validated with respect to linearity of detector response, precision of injection, specificity of chromatographic analysis, limit of detection, accuracy and precision.

The mean concentrations of Bourgeonal in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
5 consecutive days.
Frequency of treatment:
Once each day at approximately the same time each day
Details on study schedule:
n/a
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw /day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
25 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses used in this study (0, 25, 100 and 250 mg/kg/day) were selected in conjunction with the Sponsor. The known LOAEL for testicular effects in male rats for the positive control, Lilial, is 50 mg/kg/day over 5 days and the NOAEL was established as 25 mg/kg/day. Effects were clearly seen for the positive control at higher doses (100 and 200 mg/kg/day). The dose levels were therefore chosen for the test substances as those expected to cover the range where effects would be noted, without causing significant systemic toxicity.

- Rationale for animal assignment (if not random):
On arrival, the animals were removed from the transit boxes and allocated to study cages. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals.

Groups were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. Additionally, batteries of cages were rotated around the room at a weekly interval to further minimise possible spatial variations.

Each animal was assigned a number and identified uniquely within the study by a tail tattoo. Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Before the start of treatment, four males with bodyweights at the extreme of the weight range were replaced with spare animals of suitable weight from the same batch.

- Post-exposure recovery period in satellite groups: Not applicable
Positive control:
one positive control group of male rats

Examinations

Parental animals: Observations and examinations:
Bioanalysis
Analysis of urine from males treated with 100 or 25 mg/kg/day revealed the presence of the metabolite, 4-tert-butylbenzoic acid (TBBA).

Mortality
Three males receiving 250 mg/kg/day and one male receiving 100 mg/kg/day were killed for Two males (Nos. 19 and 20) treated at 250 mg/kg/day were killed for welfare reasons approximately 5 hours after administration of the first dose due to poor clinical condition.
Last in-life signs included underactivity, prostrate posture, reduced body temperature, partially closed eyelids, irregular breathing and piloerection. Macroscopic examination revealed that both animals had a dark liver with animal No. 20 also showing a distended stomach.
Male No. 23 treated at 250 mg/kg/day was killed for welfare reasons approximately 10 hours after administration of the first dose due to poor clinical condition. Last in-life signs included underactivity, hunched posture, partially closed eyelids, reduced body temperature, irregular breathing and piloerection. Macroscopic examination revealed a distended stomach.
Male No. 13 treated at 100 mg/kg/day was killed for welfare reasons approximately 12 hours after administration of the first dose due to poor clinical condition. Last in-life signs included underactivity, reduced body temperature, partially closed eyelids and piloerection.
Macroscopic examination revealed a distended stomach.
No cause of death could be established for these four animals from the tissues examined at gross or microscopic level.
Since half of the animals in the high dose Bourgeonal group had been killed, the remaining animals were killed on the morning of Day 2 of study prior to dosing.

Signs
Signs of underactivity, reduced body temperature, irregular breathing, piloerection, loose faeces and partially closed eyelids were recorded after dosing at 250 or 100 mg/kg/day; all of the signs in the 100 mg/kg/day group with the exception of underactivity were only recorded after administration of the first dose. In addition, a low incidence of prostrate or hunched posture was recorded after dosing at 250 mg/kg/day.
Signs of piloerection and loose faeces were recorded after administration the first dose of 25 mg/kg/day.
Physical examination revealed four males in the 100 mg/kg/day group with yellow faecal staining on the body.

Bodyweight
All of the three males treated at 250 mg/kg/day and killed on Day 2 of study showed bodyweight loss of 15-30 g between the start of treatment and Day 2 of study. Treatment at 100 mg/kg/day was associated with mean bodyweight loss of 14 g following administration of the first dose. This was followed by mean bodyweight stasis during Days 2-3 of study and mean bodyweight loss during Days 3-4 and 4-5 of study.
Treatment at 25 mg/kg/day was associated with mean bodyweight loss of 10 g following administration of the first dose. Thereafter, there was no clear effect of treatment on bodyweight.
As animals were deprived of food from Day 5 to Day 6, data for this period whilst included, are not commented on.

Food consumption
The males treated at 250 mg/kg/day and killed on Day 2 of study showed low food consumption between the start of treatment and Day 2 of study.
There was no clear effect of treatment at 100 or 25 mg/kg/day on mean food consumption.

Organ weights
Among animals treated at 100 mg/kg/day the mean absolute epididymal weight was marginally high and the mean testes weight was low.
There was no effect of treatment at 25 mg/kg/day.

Macropathology
Males receiving Bourgeonal at 100 mg/kg/day had enlarged epididymides (3/6), kidney depressions (2/6), thickened fore-stomach (2/6) and pale livers (5/6). Pale livers were also observed in males receiving Bourgeonal at 25 mg/kg/day (6/6).

Histopathology
Findings considered related to treatment with Bourgeonal or Lilial were seen in liver, kidney, stomach, testes and epididymides.
Generalized hepatocyte vacuolation was observed in animals treated with Bourgeonal at 100mg/kg/day (5/6 males), Bourgeonal at 25mg/kg/day (2/6 males) and with Lilial at 250mg/kg/day (6/6 males).
In the kidneys, cortical tubular vacuolation occurred in 5/6 males treated with 100mg/kg/day Bourgeonal.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. 5/6 animals treated with 100mg/kg/day Bourgeonal showed treatment-related effects in the testes and epididymides.
Oestrous cyclicity (parental animals):
n/a
Sperm parameters (parental animals):
Seminiferous tubular degeneration/atrophy, Sertoli cell vacuolation, multinucleate giant cell and luminal sloughing of spermatogenic cells in the testes and, reduced numbers of spermatozoa, sloughed germ cells in lumen and inflammation in the epididymides - 100 mg/kg/day Bourgeonal (mean urine TBBA concentration 275 μg/ml). The changes produced in the epididymides are considered secondary to their effects on the testes.
Litter observations:
n/a
Postmortem examinations (offspring):
n/a

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
not examined

Details on results (P0)

Bioanalysis
Analysis of urine from males treated with 100 or 25 mg/kg/day revealed the presence of the metabolite, 4-tert-butylbenzoic acid (TBBA).

Mortality
Three males receiving 250 mg/kg/day and one male receiving 100 mg/kg/day were killed for Two males (Nos. 19 and 20) treated at 250 mg/kg/day were killed for welfare reasons approximately 5 hours after administration of the first dose due to poor clinical condition.
Last in-life signs included underactivity, prostrate posture, reduced body temperature, partially closed eyelids, irregular breathing and piloerection. Macroscopic examination revealed that both animals had a dark liver with animal No. 20 also showing a distended stomach.
Male No. 23 treated at 250 mg/kg/day was killed for welfare reasons approximately 10 hours after administration of the first dose due to poor clinical condition. Last in-life signs included underactivity, hunched posture, partially closed eyelids, reduced body temperature, irregular breathing and piloerection. Macroscopic examination revealed a distended stomach.
Male No. 13 treated at 100 mg/kg/day was killed for welfare reasons approximately 12 hours after administration of the first dose due to poor clinical condition. Last in-life signs included underactivity, reduced body temperature, partially closed eyelids and piloerection.
Macroscopic examination revealed a distended stomach.
No cause of death could be established for these four animals from the tissues examined at gross or microscopic level.
Since half of the animals in the high dose Bourgeonal group had been killed, the remaining animals were killed on the morning of Day 2 of study prior to dosing.

Signs
Signs of underactivity, reduced body temperature, irregular breathing, piloerection, loose faeces and partially closed eyelids were recorded after dosing at 250 or 100 mg/kg/day; all of the signs in the 100 mg/kg/day group with the exception of underactivity were only recorded after administration of the first dose. In addition, a low incidence of prostrate or hunched posture was recorded after dosing at 250 mg/kg/day.
Signs of piloerection and loose faeces were recorded after administration the first dose of 25 mg/kg/day.
Physical examination revealed four males in the 100 mg/kg/day group with yellow faecal staining on the body.

Bodyweight
All of the three males treated at 250 mg/kg/day and killed on Day 2 of study showed bodyweight loss of 15-30 g between the start of treatment and Day 2 of study. Treatment at 100 mg/kg/day was associated with mean bodyweight loss of 14 g following administration of the first dose. This was followed by mean bodyweight stasis during Days 2-3 of study and mean bodyweight loss during Days 3-4 and 4-5 of study.
Treatment at 25 mg/kg/day was associated with mean bodyweight loss of 10 g following administration of the first dose. Thereafter, there was no clear effect of treatment on bodyweight.
As animals were deprived of food from Day 5 to Day 6, data for this period whilst included, are not commented on.

Food consumption
The males treated at 250 mg/kg/day and killed on Day 2 of study showed low food consumption between the start of treatment and Day 2 of study.
There was no clear effect of treatment at 100 or 25 mg/kg/day on mean food consumption.

Organ weights
Among animals treated at 100 mg/kg/day the mean absolute epididymal weight was marginally high and the mean testes weight was low.
There was no effect of treatment at 25 mg/kg/day.

Macropathology
Males receiving Bourgeonal at 100 mg/kg/day had enlarged epididymides (3/6), kidney depressions (2/6), thickened fore-stomach (2/6) and pale livers (5/6). Pale livers were also observed in males receiving Bourgeonal at 25 mg/kg/day (6/6).

Histopathology
Findings considered related to treatment with Bourgeonal or Lilial were seen in liver, kidney, stomach, testes and epididymides.
Generalized hepatocyte vacuolation was observed in animals treated with Bourgeonal at 100mg/kg/day (5/6 males), Bourgeonal at 25mg/kg/day (2/6 males) and with Lilial at 250mg/kg/day (6/6 males).
In the kidneys, cortical tubular vacu

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

n/a

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Treatment with Bourgeonal was associated with marked systemic toxicity and testicular/epididymal toxicity, and the urine contained TBBA, a known metabolite biomarker of testicular toxicity in rats. The urine of these animals did not contain TBBA. Treatment at 250 or 100 mg/kg/day Bourgeonal was not tolerated: 3/6 males and 1/6 males respectively were killed for welfare reasons after administration of the first dose due to poor clinical condition. The remaining animals in the 250 mg/kg/day group showed bodyweight loss and low mean food consumption after administration of the first dose and were killed prior to dosing on Day 2 of study. In the 250 mg/kg/day Lilial group, the marked systemic toxicity was manifest as progressive mean bodyweight loss throughout Days 1-5 of study and low mean food consumption.

Microscopic examination of tissues revealed a similar spectrum of changes following treatment with Bourgeonal or Lilial as follows:

• Generalised hepatocyte vacuolation in the liver - 100 and 25 mg/kg/day Bourgeonal and 250 mg/kg/day Lilial - Oil Red O staining of selected liver tissue sections confirmed the vacuoles contained fat. The microscopic finding of fatty vacuolation is consistent with the gross observation of pale liver tissue.

• Cortical tubular vacuolation in the kidneys - 100 mg/kg/day Bourgeonal and 250 mg/kg/day Lilial - this finding appeared to be localised to the distal tubules within the cortex.

• Epithelial hyperplasia and hyperkeratosis in the forestomach - 100 mg/kg/day Bourgeonal and 250 mg/kg/day Lilial.

• Seminiferous tubular degeneration/atrophy, Sertoli cell vacuolation, multinucleate giant cell and luminal sloughing of spermatogenic cells in the testes and, reduced numbers of spermatozoa, sloughed germ cells in lumen and inflammation in the epididymides - 100 mg/kg/day Bourgeonal (mean urine TBBA concentration 275 μg/ml).

The changes produced by both compounds in the epididymides are considered secondary to their effects on the testes.

The urine of animals treated at 25 mg/kg/day Bourgeonal also contained TBBA but at a much lower concentration (mean TBBA concentration 35.8 μg/ml) and this did not result in any testicular or epididymal toxicity.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it was concluded that treatment of sexually mature male CD rats with Bourgeonal was associated with marked systemic toxicity at 250 and 100 mg/kg/day. Treatment at 100 mg/kg/day was also associated with testicular and epididymal toxicity and the urine contained TBBA.
Executive summary:

Based on the results of this study it was concluded that treatment of sexually mature male CD rats with Bourgeonal was associated with marked systemic toxicity at 250 and 100 mg/kg/day. Treatment at 100 mg/kg/day was also associated with testicular and epididymal toxicity and the urine contained TBBA